The complete chloroplast genomes of three Alismataceae species, including the medicinally important Alisma orientale

Abstract Alismataceae is one of the early diverged families of monocotyledonous plants. We report the complete chloroplast genomes of three Alisma species, including Alisma orientale (Sam.) Juzep. 1934, A. subcordatum Raf. 1908, and A. triviale Pursh 1813, of which A. orientale is a traditional Chinese medical plant used widely to treat diuretics, diabetes, hepatitis, and inflammation. We sequenced the complete chloroplast genomes with the Illumina Nova-Seq 6000 platform using herbarium collections. The chloroplast genomes of A. orientale, A. subcordatum and A. triviale are 159,861 bp, 160,180 bp, and 159,727 bp in length, respectively. The three chloroplast genomes each contain 113 genes, including four rRNAs, 30 tRNAs genes, and 79 protein-coding genes, and the average GC content is 36.0%. Based on the whole chloroplast genomes of 19 species of Alismataceae and the close allies, the medicinally important A. orientale was found to be closely related to another medicinal plant Alisma plantago-aquatica L. 1753 in the phylogenetic analysis. The genus Alisma was supported to be monophyletic.


Introduction
Alismataceae is an ancient lineage of monocots whose complex evolutionary history has received much attention in recent years (Li et al. 2022).The genus Alisma L. is a cosmopolitan genus of aquatic and wetland plants and contains about 10 species (Bj€ orkqvist 1968;Wang et al. 2010).We report the complete chloroplast genomes of three Alisma species, A. orientale (Sam.)Juzep. 1934 from Eastern Asia, andA. subcordatum Raf. 1908, andA.triviale Pursh 1813 from North America, using herbarium collections.Of the three species we sampled, A. orientale has been valued as important in traditional Chinese medicine, and is native to Eastern and South Asia including China, Korea, Japan, Eastern Russia, India, Myanmar, Nepal, Kashmir, Mongolia, and Vietnam (Han et al. 2013).The tuber of A. orientale can be used to treat a variety of diseases, such as diuresis, oliguria, diabetes, hyperlipidemia, hepatitis, and obesity (Liu et al. 2013;Jang et al. 2015;Chinese Pharmacopoeia Commission 2020;Japanese Pharmacopoeia Commission 2021).We hope the genomic information on medicinally important species and their closely related species will lead to better taxonomic delimitations and improve resource conservation and the downstream application of medical resources, which are important for the modern utilization process of traditional natural herbs.
Total genomic DNA was extracted from herbarium specimens using the modified SDS method (Dellaporta et al. 1983;Johnson et al. 2023).An Illumina paired-end DNA library was prepared to use a KAPA HyperPrep Kit (Hoffmann-La Roche, Basel, Switzerland) and then sequenced with the Illumina Nova-Seq 6000 platform (Novogene, Sacramento, CA).
To investigate the phylogenetic relationships among the three species and their closely related taxa, whole chloroplast genomes of 19 species, including nine species of Alismataceae, nine species of other taxa of Alismatales (Araceae, Hydrocharitaceae, Zosteraceae, Tofieldiaceae, and Butomaceae) as the outgroup specie were downloaded from GenBank.The complete cp genomes were aligned with those of A. orientale, A. subcordatum, and A. triviale using MAFFT v.7.48 (Katoh et al. 2019).We constructed a maximumlikelihood (ML) tree using IQTREE v.1.6(Nguyen et al. 2015) based on the best fit model TVM þ F þ G4 and 1000 bootstrap replicates.

Results
We viewed the assembly of the chloroplast genome by mapping reads to the assembled chloroplast genome sequence using Geneious and measuring the depth of coverage (Supplementary Figure S1a-S1c).The complete genome circular assembly map was checked by Bandage v. 0.8.1 (Supplementary Figure S2a-S2c).The chloroplast genome of Alisma orientale was 159,861 bp in length, the genome of A. subcordatum was 160,180 bp in length and that of A. triviale was 159,727 bp in length (Figure 1).The sequences had a typical quadripartite structure containing a large single-copy (LSC) region, a small single-copy (SSC) region, and a pair of inverted repeat (IR) regions (Figure 2).The chloroplast genomes sequence of the three species each had 113 unique genes, including four rRNAs, 30 tRNAs genes, and 79 protein-coding genes.Among these genes, 13 cis-splicing genes were discovered.The trans-splicing gene rps12 had three unique exons (Supplementary material, Figure S3a-S3c).The overall GC content of the whole genome was 36.0%.The phylogenetic analysis showed that A. orientale clustered with Alisma plantagoaquatica L. 1753, A. triviale clustered with A. subcordatum, and Alisma was sister to the clade of Sagittaria and Caldesia.Alismataceae is sister to the Hydrocharitaceae -Butomaceae clade of Alismatales (Figure 3).

Discussion and conclusions
The whole chloroplast genomes of the three Alisma species generated in this study were consistent with the published genome structure of A. plantago-aquatica (NC_004108) (Liang et al. 2019), and the GC content was slightly different from that of A. plantago-aquatica, separated by 0.4%.These four species of Alisma formed a monophyletic group, with A. plantago-aquatica sister to the medicinally important A. orientale, and the two North America species A. subcordatum and A. triviale were sister to each other.This view was supported by the phylogenetic analysis based on the nuclear region.The close phylogenetic relationship between the medicinally important A. orientale and the widespread A. plantago-aquatica was in agreement with the Bayesian inference tree of Alisma based on chloroplast DNA loci (matK, ndhF, psbA-trnH, and rbcL) (Ito and Tanaka 2023).The sister relationship between Alismataceae and the clade of Butomaceae and Hydrocharitaceae was supported by the nuclear phylogeny (Timilsena et al. 2022).The core Alismatales consisting of Alismataceae, Hydrocharitaceae, Butomaceae, Zosteraceae, and Araceae was sister to Tofieldiaceae of the order.The higher-level phylogenetic results were largely consistent with the genus level phylogeny that sampled 10 species of Alismatales based on 79 protein-coding genes (Luo et al. 2016;Liang et al. 2019).Further studies are required to resolve the lower infrageneric relationships of the small medicinally important genus Alisma.We anticipate that our findings here will stimulate further studies on Alisma and its close relatives and contribute to the resource conservation, molecular evaluation, and identification, and the exploration and utilization of natural pharmacodynamic components of Alisma species.
Our study also shows the importance of herbarium collections in the new age of genomics and big data.Herbarium specimens are an important source for biodiversity science and can be effectively used for extracting chloroplast genome and other genomic data (herbariomics) to address questions on species delimitation and utilization of plant medicinal resources (Jiang et al. 2022;Duan et al. 2023;Wen et al. 2023).

Figure 1 .
Figure 1.Herbarium specimens used in the Alisma chloroplast genome analyses.All three specimens show complete stems, leaves, inflorescences, flowers, and the detailed information of collection and identification.Images of (A) A. orientale and (B) A. subcordatum are from the official website of the US National Herbarium (https://collections.nmnh.si.edu/search/botany/), and the use of the photos and specimens has been authorized by the Curator, Dr. Jun Wen; and (C) A. triviale from the official website of the Kathryn Kalmbach Herbarium of the Denver Botanic Gardens (https://swbiodiversity.org/seinet/collections/harvestparams.php), and the use of the photos and specimens has been authorized by the Curator Dr. Jennifer Ackerfield.

Figure 2 .
Figure 2. The chloroplast genome map of (A) A. orientale, (B) A. subcordatum, and (C) A. triviale.The map was generated by CPGView.Genes located on the inner and outer of circle are transcribed clockwise and anticlockwise, respectively.The dark grey inner circle indicates GC content.Large single-copy (LSC), small singlecopy (SSC), and inverted repeats (IRA and IRB) are indicated in the inner layer.The functional classification of the genes is provided in the bottom left corner.